This project focuses on the structure and function of adipocyte lipid storage droplets, especially the metabolism of stored triacylglycerols to release fatty acids, the primary energy reserve in animals. Intact lipid droplets were purified from differentiating 3T3-L1 adipocytes and methods were developed to purify the extremely solubilization-resistant droplet proteins, perilipin A and B, which appear to be the only major proteins located exclusively on the lipid droplet. Based on our earlier isolation of cDNAs for the rat perilipins, cDNAs for murine perilipins were isolated from a 3T3-L1 adipocyte library and full length coding sequences for perilipins A and B were obtained from a mouse genomic library; mouse and rat exhibit 93% predicted sequence identity. Further analysis of mRNAs suggests that expression of the A and B forms may be regulated independently. Lipid hydrolysis in adipocytes involves translocation of hormone- sensitive lipase (HSL) to the lipid droplet surface following elevation of cAMP which lead to phosphorylation of HSL and the perilipins. We have succeeded in eliciting in vitro an A-kinase-dependent translocation of cytosolic HSL to purified lipid droplets. A role for perilipin in lipid hydrolysis is suggested by findings in cultured adrenal cells. Y-1 adrenal cells produce steroid upon elevation of cAMP by activating cholestryl esterase, now thought to be identical to HSL of adipocytes. Western blotting and immunofluorescence reveals that perilipins A and B are associated with the lipid droplets in adrenal cells. Since perilipins are not expressed in numerous other tissues, the data strongly suggest a link between perilipin and lipid hydrolysis by HSL or HSL-like lipases. However, implicating perilipin in the maintenance of lipid droplet structure is the finding that perlipin ablation by transfection of adipoblasts with cDNA in the antisense orientation leads to an apparent dispersion of the lipid in numerous microdroplets, similar to the image produced by chronic polyphosphorylation of perilipin by A-kinase. Interestingly, over expression of perilpin by introduction of vectors bearing perilpin cDNA in the sense orientation leads to rapidly accelerated differentiation of adipocytes. Two cytokines that have been implicated in disease-associated cachexia are TNF and interleukin-6 (IL-6). We have found that both act directly on cultured adipocytes and increase the tyrosyl phosphorylation of overlapping populations of proteins. Focusing on IL-6 actions, we have established that one of the major target proteins of this cytokine is MAP kinase, which is phosphorylated and activated within minutes by physiological concentration of the cytokine.